Statistics from the American Cancer Society indicates that bladder cancer causes ˜15,000 deaths in the United States and there are ˜70,000 reported new cases annually (Siegel, Ward et al., 2011). If unchecked, bladder cancer may be lethal and the recurrent rate of bladder cancer is high. Thus early detection as well as regular monitoring for bladder cancer is recommended, ideally involving the use of non-invasive diagnostic techniques.
DNA methylation of multiple genes may contribute to the pathogenesis of bladder cancer. DNA methylation of many genes (e.g., 66 genes) has been reported in different bladder cancer patients. (See, e.g., Renard, Joniau et al., 2009). Many of the studies were performed using bladder tissues obtained from cancer patients, while others were performed using patient urine samples.
A number of different panels of genes have been reported to undergo DNA methylation associated with bladder cancer. For example, Friedrich et al. used quantitative methylation specific PCR and analyzed the apoptosis-related genes including ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB. They reported DNA methylation of DAPK, BCL2 and TERT in 78% of urine samples of high stage bladder cancer patients. (Friedrich, Weisenberger et al., 2004). Vinci et al. reported 79% of the patients with bladder cancer exhibited DNA methylation of DAPK, TERT, and BCL2. (Vinci, Giannarini et al., 2009). Hogue et al. reported DNA methylation of four (4) genes (i.e., CDKN2A, ARF, MGMT, and GSTP1) with 69% sensitivity and 100% specificity. Using a two stage analysis, Hogue et al. reported promoter DNA hypermethylation of an additional five (5) genes (i.e., APC, CDH1, RAR β2, RASSF1A, and TIMP3) on samples that were tested negative for CDKN2A, ARF, MGMT, and GSTP1, and this additional test has a sensitivity of 82% and specificity of 96%. (Hogue, Begum et al., 2006). Renard et al. identified TWIST1 and NID2 to be frequently methylated based on an arbitrary cut-off value in urine samples collected from bladder cancer patients. (Renard, Joniau et al., 2009). Costa et al. identified GDF15, HSPA2, TMEFF2, and VIM as potential DNA methylation biomarkers for bladder cancer. (Costa, Henrique et al., 2010). Reinert et al. used the microarray approach and reported potential methylated CpG sites in ZNF154, POU4F2, HOXA9, and EOMES. Chung et al. applied the microarray approach and reported six methylation markers (namely, MYO3A, CA10, SOX11, NKX6-2, PENK, and DBC1) in urine sediments of bladder cancer patients. (Chung, Bondaruk et al., 2011)
It is noteworthy that a quantitative DNA methylation assay provides a measurement of how much DNA methylation occurs (i.e., the assay provides copy numbers of the methylated gene panel). However, quantitative DNA methylation measurement is often arbitrary because the level of DNA methylation of a gene in a bladder cancer patient is in comparison to the same gene from a healthy individual. The DNA methylation level is based on an arbitrary set cut-off in those healthy individuals. Depending on the cut-off values, the quantitative assays lack consistency and contain small allowances for error in obtaining the reported sensitivities and specificities.
There still exists a continuing need in searching a panel of genes that exhibit DNA methylation associated with bladder cancer. There is also a need to utilize a non-invasive test to detect DNA methylation of such gene panel in order to serve as biomarkers in monitoring an increased risk (in occurrence) of bladder cancer. The present inventors have successfully identified the DNA methylation of a panel of BCL2, CDKN2A and NID2 genes as biomarkers of bladder cancer.